The present invention relates to novel host cells capable of over-expressing proteins at levels normally toxic thereto, and to methods of producing such bacteria and their use.
Microorganisms, and especially bacteria such as Escherichia coli, are among the most successful vehicles for over-expression of both prokaryotic and eukaryotic proteins (for reviews see Hockney, 1994: Grisshammer and Tate, 1995). However, expression systems employed to over-express such proteins are not always satisfactory. For example, over-expression of many prokaryotic proteins, including membrane proteins, some cytoplasmic proteins (Dong et al., 1995) and cell division proteins (de Boer et al., 1988; Gutzman et al., 1992) as well as the expression of toxic proteins such as DNAse (Doherty et al., 1993) is toxic to the host bacterium.
The expression of eukaryotic proteins in microorganisms can be equally problematical. Over expression of such proteins can also be toxic to the cell. Nonetheless, bacterial expression systems are used in industry and have been used to express a wide variety of proteins, including chymosin, insulin, interferons, insulin-like growth factors, antibodies including humanised antibodies, or fragments thereof. Given the widespread use of microorganisms for polypeptide expression in industry, there is a continuing need for improved expression systems.
Many different expression systems are known in the art for the expression of both endogenous and foreign proteins. In general DNA encoding the sequence of interest is contained in an expression vector, in some cases linked in-frame at the 5xe2x80x2 or 3xe2x80x2 end to another coding sequence so as to encode a fusion protein. The total coding sequence is operably linked to a promoter such that the promoter drives expression of the coding sequence. The coding sequence is also referred to herein as the xe2x80x9ctarget genexe2x80x9d.
The promoter is generally either a promoter native to the microorganism (for example the E. coli trpE promoter), a synthetic promoter such as the Tac promoter or a promoter obtainable from a heterologous organism, for example a virus, a bacterium or a bacteriophage such as phage xcex or T7 which is capable of functioning in the microorganism. The promoter may be constitutive or, more preferably, inducible. The expression vector may also contain a selectable marker gene, which may be an antibiotic resistance gene such as an ampicillin, tetracycline, chloramphenicol or kanamycin resistance gene.
Many promoter systems are available, often from commercial sources, which are suitable for expression of polypeptides in E. coli. For example, the PBAD promoter from the araBAD (arabinose) operon has been used, and shows advantageous induction properties, being inducible 1200 fold over background (Guzman et al., 1995). Others include PLAC, PTAC, PTRC, PL and PR. These and other systems are known in the literature.
Of these, one widely used promoter is PTAC (De Boer et al., 1983). This promoter is a hybrid derived from the trp and lac promoters of E. coli, and is one of the most potent E. coli based promoter systems known. It is inducible by IPTG, as with the lac promoter.
In another very widely used expression system, the target gene is transcribed from the vector by T7 RNA polymerase (Studier et at., 1990). In the E. coli BL21(DE3) host strain, used in conjunction with pET vectors, the T7 RNA polymerase is produced from the xcex-lysogen DE3 in the host bacterium, and its expression is under the control of the IPTG inducible lac UV5 promoter. This system has been employed successfully for over-production of many proteins, but in many cases significant over-production is hampered because of toxicity associated with the system (Studier et al., 1990: George et al., 1994). To date, it has widely been assumed that the toxicity was a function of the protein expressed in the expression system and that generally improved hosts would not be available (Studier et al., 1990).
The present invention surprisingly provides improved host cells which show a general improvement in tolerance of the toxic effects of expression systems.
In a first aspect, therefore, the invention provides a method for selecting host cell mutants which are resistant to expression system toxicity, comprising the steps of growing an expression system comprising host cells transformed with an expression vector, inducing the expression system such that a toxic effect is observed, and selecting viable cells in which the expression vector continues to function.
The invention also provides a host cell which is resistant to expression system toxicity.
Further aspects of the invention relate to more specific systems, for expressing particular classes of polypeptides, such as membrane proteins. Moreover, the invention provides host cells obtainable by the methods of the invention, especially bacterial host cells.
In a still further aspect of the invention, cells according to the invention may be employed in a method for the production of recombinant polypeptides which comprises transforming cells according to the invention with a vector encoding a nucleic acid sequence encoding the desired polypeptide and culturing the cells under conditions which allow for the expression of the polypeptide.
Surprisingly, the effect observed is general, in that it is observed whatever the target polypeptide which is encoded by the expression system. The cells are thus xe2x80x9cresistant to expression system toxicityxe2x80x9d, as opposed to being resistant to the expression of a particular toxic gene.
The invention in particular relates to the expression of membrane proteins, such that they either accumulate in the cytoplasm as inclusion bodies or become incorporated into the membrane system of the host and are thus easily recoverable and/or available for screening in situ.